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BioPharm April eBook: Outsourcing Resources 2019

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24 BioPharm International eBook April 2019 www.biopharminternational.com Outsourcing Resources Bioanalytical Methods Specialty assays used to under- stand the distribution, function, ef f icac y, a nd i m mu nogen ic it y of gene- and cell-therapy drugs— including cell-based neutralizing assays, qPCR, flow cytometry and enzy me-linked immunosorbent assays (ELISA)—are discussed, as well as recommended strategies for devel- opment, validation, and implemen- tation of assays for sample analysis from regulated non-clinical and clin- ical studies. Cellular and gene therapies are not new to the medical establish- ment. Cellular therapy, the transfer of live cells with a specific func- tion to a patient to treat/cure a dis- ease, was first successfully used in humans in the early 19th century in the form of blood transfusion. Bone marrow transplants, a simi- lar form of treatment, have become relatively common since the initial reported use in 1968. Gene therapy, the transfer of genetic material into specific cells to modulate gene expression or produce a new/modified protein, was first tested in a clinical trial in 1990 (1). Researchers at the National Institute of Health's Clinical Center treated two children with adenosine deami- nase deficiency using the patient's own white blood cells that had been harvested, had normal genes inserted, and were reinjected into the patient. Since 1990, due to improved u nde r st a nd i ng of t he hu ma n genome, proteome, and immune system along with improved drug delivery processes, the safety, effi- cacy, and applicability of cellular and gene therapies seem to be truly com- ing of age. Both types of therapy have been shown to cure or mitigate the under- lying cause of genetic and acquired diseases by replacing missing pro- teins or cells, modulating expression of specific proteins, and/or selec- tively killing tumor cells. Challenges remain for development of these therapies, particularly in the bioana- lytical method approach required. Questions include the following: • How much of a dosed gene ther- apy is present in circulation, in the target tissue, and how long does it stay there? • Is the target protein expression changed per the drug mechanism? • Is the vector genome released (shed) from the patient, allowing for potential transmission to other humans or the environment? • For cell therapies, do the trans- ferred cells survive and do they ex- press the appropriate markers? • Do the cells expand in vivo? The assays used to measure these characteristics are often not tradi- tional liquid chromatography–mass spectrometry (LC –MS) or ELISA used to quantify small- and large- molecule therapeutics and biomark- ers. The May 2018 FDA Bioanalytical Method Validation (BMV) Guidance for Industry (2) provides general guide- lines for all bioanalytical assays but lacks any specifics on assays that use flow cytometry, PCR, protein expres- sion/activity, or other techniques. This leaves investigators asking ques- tions about how to demonstrate the key bioanalytical attributes specified in the guidance, namely demonstrat- ing that the method measures the intended analyte, evaluating assay variability, the range in measure- ments that provide reliable data, and the effect of sample collection, han- dling, and storage on the reliability of data. GENE THERAPY ASSAYS For gene therapy programs, one of the first considerations is the biodistribution of the dosed vec- tor and genetic material (3, 4). The biodistribution assay will indicate whether the target tissue is success- fully transduced by the vector and, if so, how much is present within the tissue. The assay will also deter- mine whether other tissues beyond the specified target are affected; off target transduction will result in toxicity. It is especially important to assess whether the vector reaches the gonads and, if present, whether it is in the germ cells that can result in transmission to the next gen- eration. Sampling from biodistribu- tion studies should be performed at multiple time points and should include time of expected peak con- centrations along with multiple late time points to determine both dis- tribution/presence and clearance of the dosed gene therapy. Both the vector and transgene should be evaluated. Quantitative PCR is typically used for the biodistribution assay because of sensitivity requirements. Assay validation includes evalua- tion of intra- and inter-assay preci- sion and accuracy, specificity and selectivity, limit of detection, and stability. Expression of the gene therapy product should also be con- sidered, to help determine whether the transgene protein is expressed or if the target protein expression is altered per the dosed gene therapy. The expression may be assessed via a standard ELISA; the current 2018 FDA BMV guidance (2) provides spe- cific assay parameters to validate. These parameters include intra- and inter-assay precision and accuracy, dilutional linearity/integrity, selec- tivity, and stability, with accep- tance criteria of ± 20% bias and ≤ 20% coefficient of variation applied (25% at the lower and upper limits of quantification). In some instances, the activity of the protein may be beneficial to assess, and similar vali- dation parameters and acceptance criteria are generally straight-forward to apply. Another important assay for gene therapy programs is immunogenicity

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